In a unique integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which primary collagen defects cause skeletal fragility and other significant connective tissue symptoms and then to apply the knowledge gained from our studies to the treatment of children with these conditions. We recently focused on the development of a non-lethal animal model for OI with a classical collagen mutation. This non-lethal knock-in mouse, the Brtl mouse (Brtl), with a glycine substitution mutation in the a1(I) chain, is an excellent model for pharmacological treatment trials, for approaches to gene therapy suitable for dominant disorders, and for investigations of the skeletal matrix of OI. Our clinical studies involve children with types III and IV OI who are enrolled in age-appropriate clinical protocols for treatment and form a longitudinal study group. The OI/EDS Region of the a1(I) Collagen Chain Cabral, Letocha, Marini; in collaboration with Leikin Patients with OI/EDS form a distinct subset of osteogenesis imperfecta patients. In addition to the skeletal fragility of OI, they have characteristics of EDS, including severe laxity of large and small joints and early onset scoliosis. In 7 children with OI/EDS, we delineated mutations in the first 90 residues of the helical region of the a1(I) chain. We have determined that these collagen mutations cause abnormal N-propeptide processing, incorporation of pN-collagen into matrix and decreased diameter of dermal fibrils. This data provides a mechanism for their EDS symptoms, while the helical changes per se are responsible for bone fragility. Thus, the mechanism of their EDS is shared with patients who have EDS VIIA and B due to absence of the N-proteinase cleavage site from the a1(I) or a2(I) chain, respectively. Mutations in exon 7 resulted in helices with both decreased and normal Tm visible as a double peak on differential scanning calorimetry (DSC) of proband collagen and a broadened single peak on DSC of proband procollagen. Mutations in exons 8 to 11 showed even lower Tm of mutant helices with well separated mutant and normal DSC peaks in collagen and overlapping, but still distinct, DSC peaks in procollagen. All seven mutants had different thermal stability of collagen and procollagen, in sharp contrast to mutants beyond the first 90 amino acids whose procollagen and collagen DSC traces were identical. In vitro cleavage with N-proteinase processed only 25% of proband proa1(I) chains for exon 7 mutations, and 65-85% of proa1(I) chains for exon 8-11 mutations. The pericelluar processing of the 7 mutants was also delayed. The pN-collagen is incorporated into matrix deposited by cultured fibroblasts, with pN-a1(I) collagen prominently present in the newly incorporated and immaturely cross-linked fractions. Dermal fibrils of 6 patients were examined by electron microscopy. The fibril diameters of all 6 were significantly smaller than those of matched controls, as is seen in EDS VII. The fibril diameter was smallest for mutations in exons 7 and 8 and intermediate in size for mutations in exon 11. These assays define a folding region of a1(I) in which mutations cause a distinct OI/ED phenotype by altering the triple helical structure and secondary structure of the N-proteinase cleavage site. The retention of N-propeptide in a substantial proportion of collagen chains limits fibril diameter. The abnormal fibrils may cause laxity of joints and paraspinal ligaments directly, by reduced resistance to shearing forces, or indirectly, by altering interactions between collagen and other matrix components in the overlap zone of the D-periods. Alendronate Treatment of Brtl Mouse Marini, Uveges; in collaboration with Goldstein and Gronowicz Bisphosphonate drugs are widely administered to children with osteogenesis imperfecta (OI), but their effects on OI bone tissue containing abnormal type I collagen have not been directly examined. The Brtl mouse model for type IV OI has a glycine substitution (G349C) knocked-into one COL1A1 allele. We treated Brtl and wild type offspring of Brtl x CD-1 matings from 2-14 weeks of age with alendronate (0.219 mg/kg/wk, gift of Merck) or saline placebo. Brtl mouse weight and femoral length were significantly smaller than wild type and unchanged by alendronate. Whole bone density of femurs and lumbar vertebrae were significantly increased in both treated Brtl and wild type; treated Brtl samples attained average untreated wild type BMD. Micro CT data suggest these differences in Brtl are due to increases in bone volume rather than mineralization. Distal femoral bone volume per total volume doubled with treatment in both Brtl and wild type due to increased trabecular number. Diaphyseal cortical thickness increased by periosteal bone deposition in Brtl and wild type femurs. In treated Brtl femurs, overall geometry was reshaped to a more rounded structure. Mechanical properties of femurs were tested in 4-point bending. Alendronate treatment increased yield load, ultimate load and stiffness in both Brtl and wild type. However, alendronate failed to improve the brittleness of the Brtl femoral bone; post-yield displacement remained significantly lower in treated Brtl than wild type. In alendronate treated Brtl, fewer plump cuboidal osteoblasts were seen; many osteoblasts had an intermediate morphology with enlarged Golgi suggesting functionally exhausted cells. Our interpretation is that alendronate treatment of Brtl increases the amount of new periosteal and trabecular bone, leaving synthetically depleted osteoblasts at 14 weeks. Because of the increased bone volume post-treatment, Brtl femurs can withstand a larger load before undergoing permanent deformation. However, femur brittleness is not improved and may be exacerbated. After the yield point is reached, Brtl femurs fracture with similar additional load and deformation as if untreated. Pamidronate Treatment of Children with Types III and IV OI Letocha, Marini; in collaboration with Gerber and Paul Uncontrolled trials of bisphosphonates in OI children report increased vertebral bone density and height, improved strength and functional level and decreased fractures and bone pain. We undertook a controlled trial of pamidronate in children with Types III and IV OI. Children in the treatment group received pamidronate (10 mg/m2/day for 3 days every 3 months); all children had quarterly rehabilitation and physical therapy assessments including measurements of function, strength, and pain. Treated patients experienced a significant increase in vertebral BMD z-score compared to controls. They also had significant increases in L2 vertebral height and L1 and L2 area. In contrast to reports from uncontrolled trials, we found no significant changes in ambulation level, lower extremity strength, or pain in OI children treated with pamidronate. Motor skills related to ambulation were assessed with the 10-point BAMF. The treatment group BAMF at initiation was 6.1?1.8 vs 6.7?1.9 at 12 months (p=0.5). The control group BAMF at the initiation was 6.6?2 vs 7.02?1.31 at 12 months (p=0.61). Both groups, on average, used maximum gait aids. Manual muscle testing was assessed as the sum (total points 110) of abdominal, straight leg raise, hip abduction, extension, and flexion, and quadriceps strength. Lower extremity muscle strength did not change. The treatment group score was 67.1?20.9 at baseline vs 68.6 ? 19.7 at 12 months (p=0.88); control was 74.2?21.3 at initiation vs 74.6?14.3 at 12 months (p=0.97). There was no significant decrease in pain on a 4-point scale. Some patients reported increased endurance or decreased back pain; most patients reported no perceptible changes.